Remedy and preventive for diseases caused by NF-κB

ABSTRACT

Administration of a decoy, i.e. a compound which specifically antagonizes the nucleic acid domain to which NF-κB is bound, is effective in the treatment and prevention of diseases caused by the transcriptional regulatory factor NF-κB, such as ischemic diseases, inflammatory diseases, autoimmune diseases, cancer metastasis and invasion, and cachexia.

This application is a continuation of 09/832,841, filed Apr. 12, 2001,now abandoned, which application is a continuation of application Ser.No. 08/945,805, filed Jan. 6, 1998, now U.S. Pat. No. 6,262,033, whichapplication is the national phase of PCT/JP96/01234, filed May 10, 1996,published in a non-English language.

TECHNICAL FIELD

The present invention relates to the prevention and treatment of variousdiseases associated with NF-κB which is known to be a regulatory factorin the transcription of cytokines and adhesion factors. Moreparticularly, the invention relates to an NF-κB decoy, a compositioncomprising said decoy for the therapy and prophylaxis ofNF-κB-associated diseases, and a method for said therapy andprophylaxis.

BACKGROUND ART

A variety of diseases including asthma, cancers, heart diseases,autoimmune diseases, and viral infections manifest varying symptoms andsigns and yet it has been suggested that either an overexpression orunderexpression of one or a few proteins is a major etiologic factor inmany cases. Moreover, a variety of transcriptional regulatory factorssuch as transcription activators and transcription inhibitors areinvolved in the expression of proteins. NF-κB, a substance known to beone of such transcriptional regulatory factors, is a heterodimer of p65and p50 proteins. In the cytoplasm, NF-κB is usually present assubstance binding with IκB, an inhibition factor, and thereby preventedfrom migrating into the nucleus. However, when cell is stimulated bycytokines, ischemia, or reperfusion for whatever reason, IκB isphosphorylated and decomposed so that the NF-κB is activated andpenetrates into the nucleus. NF-κB attaches itself to the NF-κB bindingsite of the chromosome and then promotes transcription of the genelocated at downstreams. The gene controlled by NF-κB includes cytokinessuch as IL-1, IL-6, IL-8, etc. and adhesion factors such as VCAM-1,ICAM-1, etc. . .

DISCLOSURE OF THE INVENTION

Predicting that stimulation of the production of those cytokines andadhesion factors is causative of various morbidities such as ischemicdiseases, inflammatory diseases, autoimmune diseases, cancer metastasisand invation, and cachexia, the inventors of this invention did muchresearch and found that it is a rewarding therapeutic approach tosuppress expression of those genes which are activated by NF-κB byadministering a decoy of the NF-κB binding site of chromosome, that isto say a compound which specifically antagonizes the, binding site ofchromosome to which NF-κB is conjugated. The present invention has beendeveloped on the basis of the above finding.

The present invention, therefore, provides a pharmaceutical compositioncomprising an NF-κB decoy as an active ingredient for the therapy andprophylaxis of various NF-κB-associated diseases and a method for saidtherapy and prophylaxis.

The diseases in which the therapeutic/prophylactic composition of theinvention is indicated are NF-κB-associated diseases, that is to saydiseases caused by the unwanted activation of genes under control of thetranscriptional regulatory factor NF-κB, and among such diseases can bereckoned ischemic diseases, inflammatory diseases, autoimmune diseases,cancer metastasis and invasion, and cachexia. The ischemic diseaseincludes ischemic diseases of organs (e.g. ischemic heart diseases suchas myocardial infarction, acute heart failure, chronic heart failure,etc., ischemic brain diseases such as cerebral infarction, and ischemiclung diseases such as palmonary infarction), aggravation of theprognosis of organ transplantation or organ surgery (e.g. aggravation ofthe prognosis of heart transplantation, cardiac surgery, kidneytransplantation, renal surgery, liver transplantation, hepatic surgery,bone marrow transplantation, skin grafting, corneal transplantation, andlung transplantation), reperfusion disorders, and post-PTCA restenosis.The inflammatory disease mentioned above includes various inflammatorydiseases such as nephritis, hepatitis, arthritis, etc., acute renalfailure, chronic renal failure, and arteriosclerosis, among otherdiseases. The autoimmune disease mentioned above includes but is notlimited to rheumatism, multiple sclerosis, and Hashimoto's thyroiditis.Particularly the pharmaceutical composition containing the NF-κB decoyaccording to the present invention as an active ingredient is verysuited for the therapy and prophylaxis of reperfusion disorders inischemic diseases, aggravation of the prognosis of organ transplantationor organ surgery, post-PTCA restenosis, cancer metastasis and invasion,and cachexia such as weight loss following the onset of a cancer.

The NF-κB decoy that can be used in the present invention may be anycompound that specifically antagonizes the NF-κB binding site of thechromosome and includes but is not limited to nucleic acids and theiranalogs. As preferred examples of said NF-κB decoy, there can bementioned oligonucleotides containing the nucleotide sequence ofGGGATTTCCC (the sequence from the 8th through the 17th nucleotides fromthe 5′-end of SEQ ID NO: 1 in Sequence Listing) or its complementarysequence, muteins thereof, and compounds containing any of them withinthe molecule. The oligonucleotides may be DNAs or RNAs, and may containmodified nucleotides and/or pseudonucleotides. Furthermore, thoseoligonucleotides, variants thereof, or compounds containing any of themmay be single-stranded or double-stranded and linear or cyclic. Thevariants are those involving mutations such as substitution, additionand/or deletion of any part of the above-mentioned sequence, and meannucleic acids specifically antagonizing the binding site of chromosometo which NF-κB is conjugated. The more preferred NF-κB decoy includesdouble-stranded oligonucleotides each containing one or a plurality ofthe above nucleotide sequence and variants thereof. The oligonucleotidewhich can be used in the present invention includes oligonucleotidesmodified so as to be less susceptible to biodegradation, such as thoseoligonucleotides containing the thiophosphoric diester bond availableupon substitution of sulfur for the oxygen of the phosphoric diestermoiety (S-oligo) and those oligonucleotides available upon substitutionof a methyl phosphate group carrying no electric charge for thephosphoric diester moiety.

Regarding to a technology for producing the NF-κB decoy for use in thepresent invention, the conventional chemical or biochemical methods forsynthesis can be utilized. When a nucleic acid, for instance, is to beused as the NF-κB decoy, the methods for nucleic acid synthesis whichare commonly used in genetic engineering can be employed. For example,the objective decoy oligonucleotide can be directly synthesized on a DNAsynthesizer. Or a nucleic acid or its fragments, each synthesizedbeforehand, can be amplified by PCR or using a cloning vector or thelike. Furthermore, the desired nucleic acid can be obtained by suchprocedures as cleavage with restriction enzymes or the like and/orligation by means of DNA ligase or the like. In order to obtain a decoynucleotide which is more stable within cells, the base, sugar or/andphosphoric acid moieties of the nucleic acid may be alkylated, acylated,or otherwise chemically modified.

The pharmaceutical composition containing the NF-κB decoy as an activeingredient according to the present invention is not limited in formonly if the active ingredient may be taken up by the cells in theaffected site or the cells of the target tissue. Thus, the NF-κB decoy,either alone or in admixture with the common pharmaceutical carrier, canbe administered orally, parenterally, topically or externally. Thepharmaceutical composition may be provided in liquid dosage forms suchas solutions, suspensions, syrups, liposomes, lotions, etc. or in soliddosage forms such as tablets, granules, powders, and capsules. Wherenecessary, those pharmaceutical compositions may be supplemented withvarious vehicles, excipients, stabilizers, lubricants, and/or otherconventional pharmaceutical additives, such as lactose, citric acid,tartaric acid, stearic acid, magnesium stearate, terra alba, sucrose,cornstarch, talc, gelatin, agar, pectin, peanut oil, olive oil, caccaobutter, ethylene glycol, and so on.

Particularly when a nucleic acid or a modification product thereof isused as the NF-κB decoy, the preferred dosage form includes those whichare generally used in gene therapy, such as liposomes inclusive ofmembrane fusion liposomes utilizing Sendai virus and liposomes utilizingendocytosis, preparations containing cationic lipids such asLipofectamine (Life Tech Oriental) or virosomes utilizing a retrovirusvector, adenovirus vector, or the like. Particularly preferred aremembrane fusion liposomes.

The structure of such a liposomal preparation may be any of a largeunilamellar vesicle (LUV), a multi-lamellar vesicle (MLV), and a smallunilamellar vesicle (SUV). The approximate size of vesicles may rangefrom 200 to 1000 nm for LUV, from 400 to 3500 nm for MLV, and from 20 to50 nm for SUV but in the case of a membrane fusion liposomal preparationusing Sendai virus, for instance, MLV with a vesicular system of200-1000 nm in diameter is preferably employed.

There is no limitation on the technology for liposome production only ifthe decoy can be successfully entrapped in vesicles. Thus, suchliposomes can be manufactured by the conventional techniques such as thereversed phase evaporation method (Szoka, F., et al: Biochim. Biophys.Acta, Vol. 601 559 (1980)), ether injection method (Deamer, D. W.: Ann.N.Y. Acad. Sci., Vol. 308 250 (1978)), and surfactant method (Brunner,J., et al: Biochim. Biophys. Acta, Vol. 455 322 (1976)), to name but afew examples.

The lipid that can be used for constructing a liposomal structureincludes phospholipids, cholesterol and its derivatives, andnitrogen-containing lipids but phospholipids are generally preferred.The phospholipid that can be used includes naturally-occurringphospholipids such as phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin,soybean lecithin, lysolecithin, etc., the corresponding phospholipidshydrogenated by the conventional method, and synthetic phospholipidssuch as dicetyl phosphate, distearoylphosphatidylcholine,dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine,dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine,eleostearoylphosphatidylethanolamine, eleostearoylphosphatidylserine,and so on.

The lipids inclusive of phospholipids can be used each alone or in asuitable combination. By using a lipid containing a positively-chargedatomic group such as ethanolamine or choline within the molecule, thebinding rate of an electrically negative decoy nucleotide can beenhanced. In addition to the principal phospholipid, various compoundssuch as cholesterol and its derivatives, stearylamine, -tocopherol,etc., which are known as liposome additives, can be added in themanufacture of liposomes.

To the resulting liposomes can be added a membrane fusion promoter suchas Sendai virus, inactivated Sendai virus, a membrane fusion promotingprotein purified from Sendai virus, polyethylene glycol, or the like canbe added for assisting in the intracellular uptake by the cells at theaffected site or of the target tissue.

A typical procedure for the production of pharmaceutical liposomes isnow described in detail. The above-mentioned liposome-forming substanceas well as cholesterol or the like is dissolved in an organic solventsuch as tetrahydrofuran, chloroform, ethanol, or the like. In a suitablevessel, the solvent is distilled off under reduced pressure to leave afilm of the liposome-forming substance on the inside wall of the vessel.Then, a buffer containing the NF-κB decoy is added and the mixture isstirred. After optional addition of said membrane fusion promoter, theliposomes are isolated. The liposomes in which the NF-κB decoy has thusbeen entrapped are suspended in a suitable medium or a lyophilizatethereof is redispersed in a suitable medium for use in therapy. Themembrane fusion promoter may be added in the interim period afterisolation of the liposomes and before use.

There is no limitation on the decoy content of the pharmaceuticalcomposition containing the NF-κB decoy as an active ingredient only ifthe decoy is contained in amounts effective to control NF-κB-associateddiseases. Thus, the decoy content can be liberally selected according tothe disease to be controlled, the target site, dosage form, and dosageschedule.

The pharmaceutical composition containing the NF-κB decoy as an activeingredient as provided in the above manner can be administered byvarious methods according to the type of disease and the kind of decoycontained. Taking ischemic diseases, inflammatory diseases, autoimmunediseases, cancer metastasis or invasion, and cachexia as examples, thecomposition can be infused intravascularly, applied directly to theaffected area, injected into the lesion, or administered into theregional blood vessel in the affected region. As a further specificexample, when PTCA is performed for infarction of an organ, thepharmaceutical composition can be administered into the local bloodvessel concurrently with the operation or pre- and postoperatively. Fororgan transplantation, the graft material can be previously treated withthe composition of the invention. Furthermore, in the treatment ofosteoarthritis or rheumatism, the composition can be directly injectedinto the joint.

The dosage of the NF-κB decoy is selected with reference to thepatient's age and other factors, type of disease, the kind of decoyused, etc. but for intravascular, intramuscular, or intraarticularadministration, for instance, a unit dose of 10-10,000 nmoles cangenerally be administered once to a few times daily.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are intended to describe the present invention infurther detail.

EXAMPLE 1

Synthesis of an NF-κB Decoy (Decoy Oligonucleotide)

On a DNA synthesizer, an NF-κB decoy oligonucleotide and a scrambleddecoy oligonucleotide (an oligonucleotide having the same basecomposition as the NF-κB decoy oligonucleotide but a randomizedsequence), the nucleotide sequences of which are shown below, wererespectively synthesized from S-oligonucleotides. Those nucleotides wereheated at 80° C. for 30 minutes and then allowed to cool to roomtemperature over 2 hours to provide double-stranded DNAs.

NF-κB decoy oligonucleotide

CCTTGAAGGGATTTCCCTCC (SEQ ID NO: 1) GGAACTTCCCTAAAGGGAGG (SEQ ID NO: 3)

Scrambled decoy oligonucleotide

TTGCCGTACCTGACTTAGCC (SEQ ID NO: 2) AACGGCATGGACTGAATCGG (SEQ ID NO: 4)

EXAMPLE 2

Production of Liposomal Preparations

Phosphatidylserine, phosphatidylcholine, and cholesterol, provided in aweight ratio of 1:4.8:2 (a total of 10 mg), were dissolved intetrahydrofuran. Using a rotary evaporator, the tetrahydrofuran wasremoved from the lipid solution to leave the lipid in the form of a filmadherent to the flask wall. To this was added 200 ml of saline (BSS; 139mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6) containing the NF-κB decoyoligonucleotide (0.7 mg) prepared in Example 1 and the mixture wasstirred and sonicated under the usual conditions to provide a suspensionof liposomes containing the NF-κB decoy oligonucleotide. This suspensionof liposome vesicles (0.5 ml, lipid content 10 mg) was mixed withpurified Sendai virus (Z strain, 10000 hemaglutinating units) exposed toUV radiation (110 erg/mm²/sec) 3 minutes before use and the mixture wasmade up to 4 ml with BSS. This mixture was held at 4° C. for 5 minutesand, then, subjected to gentle shaking at 37° C. for 30 minutes. Afterthe Sendai virus not bound to the liposomes was removed by sucrosedensity gradient centrifugation, the uppermost layer was separated andits concentration was adjusted with BSS to provide a liposomalpreparation containing 8 μM NF-κB decoy oligonucleotide as entrapped. Aliposomal preparation was similarly produced using the scrambled decoyoligonucleotide of Example 1 in lieu of the NF-κB decoy oligonucleotide.

EXAMPLE 3

A Reperfusion Model Experiment

(1) Method

After 9˜10-week-old SD rats were anesthetized with pentobarbital sodium,a cannula was inserted into the left carotid artery adjacent to theairway and indwelled near the aortic valve of the heart (close to theostium of the coronary artery). In addition, the trachea was cannulatedand the animal was placed on supportive respiration by connecting thetracheal cannula to an artificial respirator. Thereafter, a leftintercostal incision was made and the left descending anterior branch ofthe rat heart was ligated to produce ischemia. After 30 minutes, theligating suture was cut to start reperfusion. Immediately thereafter,1.5 ml/rat of the liposomally entrapped NF-κB decoy nucleotide orscrambled decoy nucleotide prepared in Example 2 was administered viathe cannula indwelled close to the ostium of the coronary artery. Afterthe chest was closed, the trachea was also sutured and the animal waskept alive. After 24 hours, the rat was reanesthetized and the heart wasenucleated and washed with saline. The ventricle of the rat heart wassliced into six sections which were stained with tetrazolium chloride(TTC). The six sections were respectively photographed and subjected toimage analysis. The infarcted area was calculated by means of thefollowing equation.Infarction rate (%)=the sum of infarct areas of 6 sections/the sum of areas of 6sections×100

Statistical analysis was made by multiple comparison (ANOVA).

(2) Results

The results are presented in Table 1. In the untreated control group andthe scrambled decoy treatment group, myocardial infarcts were found inapproximately equal degrees. In the group given the NF-κB decoynucleotide, the infarct was suppressed to 19% with a significantdifference (P<0.01) from the untreated group and the scrambled decoygroup.

TABLE 1 NF-κB decoy Scrambled nucleotide decoy Untreated group groupgroup Myocardial 19 2% 28 1% 28 1% infarct area/ total area

A similar inhibitory effect was found when the liposomes wereadministered immediately before induction of infarction.

EXAMPLE 4

Inhibition of Cancer Metastasis

(1) Method

To 7-week-old female mice of the C57BL/6 strain, 1×10⁴ murine reticulumcell sarcoma M5076 cells were administered intravenously and 24 hourslater 0.2 ml (6 nmoles) of an NF-κB decoy nucleotide prepared in thesame manner as Example 2 was administered intravenously. A control groupreceived 0.2 ml of saline in the same manner. On day 14 afterintravenous administration of M5076, the animal was autopsied and thenumber of tumor nodules on the surface of the liver was counted underthe stereoscopic microscope. Each group consisted of 10 mice. Forstatistical analyses, Kruskal-Wallis test and Dunnett's multiplecomparison were used.

(2) Results

Whereas the mean number of tumor nodules in the control group was 166with a median value of 173 (116-198), the NF-κB decoy treatment groupshowed a mean number of 29 and a median number of 27 (19-54). Thus,between the NF-κB decoy treatment group and the control group, asignificant difference was found at the 1% level.

EXAMPLE 5

Inhibition of Cachexia

(1) Method

Using 7-week-old male BALB/c mice, a 2 mm cubic tumor mass of murinecolon cancer line Colon 26 was transplanted subdermally. Beginning day 7after transplantation, 0.2 ml (6 nmoles) of the NF-κB decoy or thescrambled decoy was administered into the tumor mass and the body weightand tumor weight were serially determined. The animal was autopsied onday 13 and the epididymal fat and gastrocnemius muscle were isolated andweighed. Furthermore, the wet carcass weight exclusive of all theremaining organs and tumor was determined. The tumor weight wascalculated from the major and minor diameters of each tumor mass bymeans of the following equation.Tumor weight (mg)=major diameter×minor diameter²/2

Each group consisted of 10 mice. Statistical analyses were made by ANOVAin one-way layout and Dunnett's multiple comparison.

(2) Results

In the tumor-bearing group, growth of the tumor resulted in significantdecreases in body weight, epididymal fat weight, gastrocnemius muscleweight, and wet carcass weight. In the NF-κB decoy group, improvementswere obtained, amounting to 47% for body weight, 42% for epididymal fatweight, 60% for gastrocnemius weight, and 52% for wet carcass weight.However, no improvement was found in the scrambled decoy group. Therewas no definite effect on tumor weight.

1. A method of treating or preventing an NF-κB-associated disease whichcomprises administering to a human an effective amount of apharmaceutical composition comprising a polynucleotide NF-κB chromosomalbinding site decoy which antagonizes NF-κB-mediated transcription of agene located downstream of said binding site, wherein the polynucleotidecomprises nucleotides 8-17 of SEQ ID NO:1.
 2. A method for treatment ofan NF-κB-associated which comprises administering to an animal aneffective amount of a polynucleotide NF-κB chromosomal binding sitedecoy which antagonizes NF-κB-mediated transcription of a gene locateddownstream of a NF-κB binding sit; wherein said polynucleotide comprisesnucleotides 8-17 of SEQ ID NO:1.
 3. The method according to claim 2wherein the NF-κB-associated disease is selected from the groupconsisting of: an ischemic disease, an inflammatory disease, and anautoimmune disease.
 4. The method according to claim 2 wherein theNF-κB-associated disease is an ischemic disease.
 5. The method accordingto claim 2 wherein the NF-κB-associated disease is selected from thegroup consisting of: a reperfusion disorder in ischemic disease,aggravation of a prognosis of an organ transplantation, aggravation of aprognosis of an organ surgery, and a post-PTCA restinosis.
 6. The methodaccording to claim 2 wherein the NF-κB-associated disease is selectedfrom the group consisting of: a reperfusion disorder in ischemic heartdisease, aggravation of a prognosis of a heart transplantation,aggravation of a prognosis of a heart surgery, and post PTCA restinosis.7. The method according to claim 2 wherein the NF-κB-associated diseaseis selected from the group consisting of: a cancer metastasis, a cancerinvasion, and cachexia.
 8. The method according to claim 2, wherein thepolynucleotide consists essentially of nucleotides 8-17 of SEQ ID NO:1.9. The method according to claim 1, wherein the NF-κB-associated diseaseis selected from the group consisting of: an ischemic disease, aninflammatory disease, and an autoimmune disease.
 10. The methodaccording to claim 1, wherein the NF-κB-associated disease is anischemic disease.
 11. The method according to claim 1, wherein theNF-κB-associated disease is selected from the group consisting of: areperfusion disorder in ischemic disease, aggravation of a prognosis ofan organ transplantation, aggravation of a prognosis of an organsurgery, and a post-PTCA restinosis.
 12. The method according to claim1, wherein the NF-κKB-associated disease is selected from the groupconsisting of: a reperfusion disorder in ischemic heart disease,aggravation of a prognosis of a heart transplantation, aggravation of aprognosis of a heart surgery, and post PTCA restinosis.
 13. The methodaccording to claim 1, wherein the NF-κB-associated disease is selectedfrom the group consisting of: a cancer metastasis, a cancer invasion,and cachexia.
 14. The method according to claim 1, wherein thepolynucleotide consists essentially of nucleotides 8-17 of SEQ ID NO:1.